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Branched-chain polyamine stabilizes RNA polymerase at elevated temperatures in hyperthermophiles
Yuka Yamori1, Masafumi Hamakawa1, Ryota Hidese1
1Department of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, 669-1337, Hyogo, Japan.
Amino Acids
|May 19, 2019
Summary
Branched-chain polyamines (BCPAs) are crucial for thermophile growth and gene expression. BCPAs enhance RNA polymerase activity and stability at high temperatures, improving transcription-translation coupling.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Branched-chain polyamines (BCPAs) are unique polycations found in thermophilic organisms.
- Thermococcus kodakarensis, a hyperthermophile, synthesizes N4-bis(aminopropyl)spermidine via BCPA synthase A (BpsA).
- A bpsA deletion mutant showed impaired growth at high temperatures, suggesting BCPA's role in thermotolerance and gene expression.
Purpose of the Study:
- To investigate the role of BCPAs in gene expression and RNA polymerase (RNAP) function in Thermococcus kodakarensis.
- To compare the enzymatic properties of RNAP from wild-type (WT) and BCPAs-deficient mutant cells.
- To elucidate the mechanism by which BCPAs influence transcription and translation at high temperatures.
Main Methods:
- Extraction and purification of RNA polymerase (RNAP) core fractions from WT and bpsA deletion mutant (DBP4) T. kodakarensis strains.
- LC-MS analysis to identify associated proteins, including ribosomal proteins, in RNAP complexes.
- In vitro transcription assays to measure RNAP activity at different temperatures and in the presence of spermidine (SPD) and BCPAs.
- Thermal stability assays to assess the protective effect of BCPAs on RNAP.
Main Results:
- RNAP from WT cells contained nine ribosomal proteins, while RNAP from the mutant (DBP4) contained only one, indicating BCPAs facilitate RNAP-ribosome linkage.
- BCPAs significantly enhanced the transcriptional activity of RNAPDBP4 at lower concentrations compared to SPD.
- BCPAs protected RNAPDBP4 from thermal inactivation at 90°C, suggesting a stabilizing effect.
Conclusions:
- BCPAs are essential for efficient coupling of transcription and translation in T. kodakarensis by promoting RNAP-ribosome interactions.
- BCPAs enhance RNAP transcriptional activity and stability at high temperatures, contributing to thermotolerance.
- BCPAs play a critical role in regulating gene expression in hyperthermophiles.
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